Abstract Addiction is a learning and memory disorder, wherein conditioned responses to seek drug lead to chronic relapse. These conditioned responses can be triggered by exposure to reminder cues associated with the drug, and they can be inhibited through a process called extinction. The prefrontal cortex, especially the infralimbic(IL)subregioninrodents,iscommonlyrecruitedduringextinctionlearningtoexertinhibitorycontrol overbehavior.Thisistrueforanumberofbehaviors,includingconditionedcocaineseeking,foodseeking,and fear.However,preliminarydatasuggestthatthisregionisnotrecruitedduringextinctionofheroinseeking,an observation consistent with numerous reports suggesting the role of IL cortex may be fundamentally different for heroin seeking. Alternatively, there may be distinct neuronal ensembles within IL that drive versus inhibit drug seeking. The anatomical connections of IL include three major efferent targets known to regulate drug seeking, namely the lateral hypothalamus, the nucleus accumbens shell, and the basolateral amygdala. We proposethatILneuronalensemblesprojectingtodistincttargetswilldriveversusinhibitheroinversuscocaine seeking, respectively. Aim 1 of this NIDA R21 proposal will determine whether functional neuronal ensembles exist within IL that are capable of driving versus inhibiting heroin and cocaine seeking. Fos-activated neurons will be tagged with designer receptors (TRAP-DREADDs) and subsequently reactivated using the designer drug, clozapine-N-oxide. This chemogenetic form of memory retrieval will be used to examine the role of the original drug-cue memory versus extinction memory on relapse. Aim 2 of this proposal will examine the efferenttargetsoftheseensemblesusingtheretrogradetracerAAVrg-GFP.Theseexperimentswilldetermine whethertheinhibitoryfunctionofIListrulyabsentforheroinseeking,orwhetherithasbeenoverlookeddueto the use of inadequate methods targeting all IL neurons indiscriminately. Furthermore, this work will provide new insight into the specific neuroanatomy of drug memory engrams and the extent to which they can be exploitedtoreducerelapse.